The Activation Of TLR4 Mediated MyD88 Dependent Signal Pathway In Oxygen-glucose Deprivation Treated PC12 Cells

IntroductionCerebral ischemia followed by reperfusion is the leading cause of morbidity and mortality. However, the mechanism involved in this process remains elusive. Now increasing number of reports have suggested that the neuroinflammatory response induced by activation of innate immunity plays a key role in the ischemia reperfusion induced injury.TLR4 functions as a receptor for lipopolysaccharide (LPS), which is a glycolipid component of the outer membrane of Gram-negative bacteria. Besides LPS, endogenous ligands such as high-mobility group box 1 (HMGB1), heat-shock proteins 60 (HSP60) released from damaged cells can also signal through TLR4 and result in the activation of NF-κB and production of numerous proinfiammatory mediators such as TNF-a, IL-1p and IL-6 which contribute to local inflammation.Moreover, the expression of TLR4 in central nervous system (CNS) is not just limited to immune cells and glial cells, such as microglias, astrocytes and oligodentrocytes. Recent findings have indicated that cortical neurons also express TLR4 and the activation of signaling pathway through TLR4 in neurons renders them vulnerable to ischemia induced death.The activation of TLR4 leads to the recruitment of TIR domain-containing proteins such as MyD88 and TIR domain-containing adaptor protein inducing interferon-β(TRIF), for that reason the signal pathway through TLR4 can be classified into MyD88-dependent and MyD88-indepent/TRIF-dpendent signaling pathway.MyD88 originally isolated as one of the 12 myeloid differentiation primary response genes, is an adaptor protein that is critical for transducing signals from all 10 TLR family members. The adaptor protein MyD88 possesses a C-terminal that interacts with TLR and IL-1R TIR domains and the N-terminal death domains which can bind to interleukin-1 receptor-associated kinase (IRAK) family. Studies have shown that mice genetically deficient in adaptor molecule MyD88 were protected from dysfunction and histological damage in kidney and myocardial IRI. These results suggest that signaling via MyD88 is the dominant pathway leading to ischemia reperfusion injury. Nevertheless, some reports suggested that TLR4 activation mediates liver IRI inflammatory response via IFN regulatory factor3-Dependent MyD88-Independent pathway. Based on that, the exact role of MyD88 played in ischemia reperfusion injury is still elusive. Different from previous study, we used neuronal cells subjected to OGD as an in vitro ischemic injury model. We investigated whether MyD88-dependent pathway plays a role in the IRI pathogenesis by blocking MyD88-dependent signaling pathway using RNA interference (RNAi).Material and methods1. Antibody and ReagentThe polyclonal anti-TLR4, anti-MyD88 and monoclonal mouse against (3-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, USA) The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dimethyl sulfoxide (DMSO) and propidium iodide(PI) were obtained from Sigma(Sigma, USA) and all other reagents were purchased from Sigma unless mentioned.2. Cell culture and Oxygen-Glucose DeprivationRat pheochromocytoma PC 12 cells were maintained in RPMI 1640 medium (Gibico, USA) supplemented with 5% fetal bovine serum (FBS),10% horse serum (Sigma. USA),100μg/ml streptomycin and 100U/ml penicillin on poly-L-lysine-coated dishes at 37℃in a humidified atmosphere of 5% CO2 and 95% air. And OGD model for PC 12 Cells was established.3. The expression of TLR4 and MyD88 protein in OGD treated PC12cells were detected by Western Blotting in different group.4. The recombinant virus was packaged using Lentiviral vector Expression Systems (Shanghai, GeneChem). PC12 cells were transfected by Enhanced Infection Solution. For knockdown efficiency test, cells were harvested 96 hours later and lysed for Western blotting analysis. One of the most efficient short hairpin RNA (shRNA) sequence (19-mer sequence,5'-CATACGCAACCAGCAGAAA-3') was used in functional assays in this paper.5. The expression of TLR4 was evaluated by RT-PCR. The expression level of IL-1βand TNF-a were quantified with real-time PCR by using SYBR PrimscripTM RT-PCR kit (TaKaRa, China) in an ABI Prism 7500 sequence detection system.6. TNF-a were measured in culture supernatant using ELISA kit according to the manufacturer's instructions.7. Cell death was determined by PI and Hoechst 33342 double fluorescent staining.8. Cellular viability was evaluated by the reduction of MTT to formazan.9. Statistical analysisAll results are expressed as mean±standard deviation. Data were analyzed using One-way ANOVA for multiple comparisons. The level of statistical significance was set at P<0.05 and P<0.01.Results1. The expression of TLR4 in PC 12 cells was up-regulated in cells exposed to OGDWe used RT-PCR and western blot to reveal the presence of TLR4 in PC12 cell line and the expression of TLR4 was increased at both mRNA(Fig.1)and protein level(Fig.2A and B) when the cells were treated with OGD. The protein of TLR4 began to increase after 2h OGD treatment followed by 24 hour reperfusion and the maximal 3.5-fold (p<0.01) increase was observed in 3h OGD treatment group. After 5h treatment of OGD, the expression of TLR4 was reduced to a level similar to that under 2h OGD., but still significantly increased compared with control group (p<0.05)The change of mRNA level was consistent with the change of protein which reached the peak after 3h OGD up to 3 fold increase compared with control group(P＜0.01).2. The expression of MyD88 was increased in respond to OGD insultData from western blot analysis showed that level of MyD88 was increased significantly for 3h (p<0.01) and 4h (p<0.01) after OGD treatment (Fig2 A and C). However, expression of MyD88 after 5h OGD treatment didn't show significant increase compared with the control group. MyD88 expression peaked after 3h OGD, reaching about 3 fold increase compared with control group. Then the level of MyD88 decreased to about 2 fold compared with the control group. The expression of MyD88 dropped to similar to control after 5h OGD treatment.3. Knockdown of MyD88 using lentivirus vector based RNAiLentiviral expression vectors containing enhanced green fluorescence protein (EGFP) and targeting sequence against MyD88 (RNAi group), or vector that contains negative control (NC) sequence were constructed. After the transfection, the endogenous MyD88 protein was assessed by western blot. The expression of MyD88 in RNAi group was reduced 80±11%(P<0.01).4. MyD88-dependent signal pathway is required for the production of IL-1βand TNF-αIn the present study, we found that both the protein and mRNA of TNF-αwere elevated when PC12 cells were treated with OGD for 3h. Using RNAi to inhibit the expression of MyD88, our result indicated that RNAi group showed a significant decrease of TNF-α, either compared with OGD group or NC group(p<0.05). Furthermore, semi-quantitative real-time PCR revealed that mRNA of IL-1(3 also increased significantly in OGD group and NC group compared with control group. On the other hand, RNAi group exhibits significant reduction of IL-1βcompared with OGD and NC group.5. Knockdown of MyD88 promotes cell viability in OGD treated PC 12 cellsWe used MTT to evaluate the cell viability after OGD. Our results indicate that after 3h OGD treatment and 24h reperfusion, cell viability was reduced to 52.79±5.76%in OGD group, and 49.97±3.45%in negative virus transfection group (NC), also there is no significant difference between these two groups. Nevertheless, viability of RNAi group was reduced to 65.12±1.55%, significantly increased when compared to OGD and NC group (p<0.01). The apoptotic and necrotic cells were detected by Hoechst 33342/PI staining, after 3h OGD treatment, almost no necrotic cells was detected. The apoptotic cells of RNAi group were markedly reduced to 12.7±0.8%, while the percentage of apoptotic cells of OGD group was 24.7±6.42%and the NC group was 26.2±5.7%(P<0.05 RNAi vs. control and P<0.01 vs. NC, n=3).DiscussionTLRs have been identified in the neuron and are thought to play an important role in the brain's response to tissue injury, including trauma, hemorrhagic shock, and ischemia. Recent evidences indicate that during the first hours of stroke, TLR4 signaling plays a pivotal role in the triggering of neuronal death. Our data demonstrate the expression of TLR4 in PC12 cells, and that TLR4 and MyD88 were up-regulated and activated in the PC12 cells treated with OGD. The expression of TLR4 was in time-dependent pattern at both mRNA and protein level. Meanwhile, MyD88 which is a downstream adaptor protein of TLRs, was also increased significantly after 3-4h OGD treatment. The expression of MyD88 is consistent with the expression change of TLR4 during the OGD insult. Taken together, we conclude that TLR4 is activated and mediates the signaling through MyD88 in PC12 cells in respond to OGD.Endogenous ligands such as HSP70, HMGB1 were found to activate TLR2 and TLR4, initiating an inflammatory response in the absence of pathogens. Previous reports have shown that inhibition of OGD-induced HMGB1 release could attenuate the PC 12 cell death in ischemia injury. HMGB1 levels are increased by ischemia/reperfusion in the liver and activation of the innate immune system by HMGB1 requires TLR4-dependent signaling. However, the exact ligand that activated TLR4 in PC 12 cells treated with OGD is still unknown.Increasing number of evidence suggest that anti-inflammation treatment could ameliorate ischemia damage. The MyD88 adapter protein links members of the toll-like receptor (TLR) to the downstream activation of Nuclear Factor-KB, which induces the production of proinflammatory cytokine IL-1β, TNF-a and IL-6. These cytokines exacerbate inflammatory response in organs, induce cell apoptosis and cause more damage to the ischemic organs. Previous reports have shown that exogenous TNF-a exacerbates focal ischemic injury and blocking endogenous TNF-a in a stroke can be expected to reduce focal ischemic injury. In the present study, we found that IL-βand TNF-a level were elevated when PC 12 cells were treated with OGD. We also observed that level of TNF-a and IL-1(3 were decreased when MyD88 expression was suppressed by RNAi.According to the recent report, TLR4 mediated energy deprivation induced activation of the JNK-AP1 pathway and Caspase-3 in cortical neurons. In our study MyD88 knockdown group exhibited significantly decreased apoptotic cells when compared with OGD for 3h group. This may be concerned with the decreased production of proinflammatory cytokines and blocked signaling to JNK-AP1. Therefore, TLR4 mediated MyD88-dependent signal pathway may play a vital role in ischemia induced cell death.Conclusion1. In the present study, we identified the expression of TLR4 in PC12 cell line for the first time. And the expression of TLR4 was up-regulated in PC12 cells subjected to OGD.2. The adaptor protein MyD88 was activated in PC12 cells treated with OGD.3. RNAi silencing the expression of MyD88 decreased the production of IL-1βand TNF-a, promoted the cell viability and decreased the percentage of apoptotic cells.